Use of inhaled NO as anti-inflammatory agent

ABSTRACT

A method for lessening or preventing non-pulmonary ischemia-reperfusion injury or inflammation in a mammal by identifying a mammal which has ischemia-reperfusion or is at risk for developing ischemia-reperfusion in a non-pulmonary tissue; and causing the mammal to inhale a therapeutically effective amount of gaseous nitric oxide sufficient to diminish the ability of leukocytes or platelets to become activated in a manner that contributes to an inflammatory process at the site of the ischemia-reperfusion or inflammation in the non-pulmonary tissue, thereby lessening or preventing non-pulmonary ischemia-reperfusion injury in the mammal.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit from provisional applicationSerial No. 60/062,926 filed Oct. 21, 1997.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] Work on this invention was supported, in part, with funds fromthe United States government (USPHS grants HL66377, HL42397, andHL45895). The government therefore has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The field of the invention is treatment of ischemia-reperfusioninjury and inflammation.

BACKGROUND OF THE INVENTION

[0004] Nitric oxide (NO) is a cell membrane-permeable, free radicalmolecule which accounts for the vasodilator activity ofendothelium-derived relaxing factor (reviewed in Schmidt et al., Cell78:919-925 [1994]). NO interacts with several intracellular moleculartargets, one of which is soluble guanylate cyclase (sGC). Binding of NOto the heme group in sGC stimulates the conversion of guanosinetriphosphate (GTP) to guanosine-3′,5′-cyclic monophosphate (cGMP). cGMPexerts it effects on cells, in part, through its action oncGMP-dependent protein kinase (cGDPK). Additional cGMP targets includecGMP-gated ion channels and cGMP-regulated cyclic nucleotidephosphodiesterases. Phosphodiesterases (PDEs) inactivate cGMP byconverting it to GMP. At least four types of PDEs appear to participatein the metabolism of cyclic nucleotides in non-ocular tissues (types 1-3and 5), only one of which, type 5 (PDE5), is specific for cGMPmetabolism. Several agents act as selective inhibitors of PDE5,including dipyridamole and Zaprinast™.

[0005] The biological effects of NO are also mediated bycGMP-independent mechanisms. NO can serve as an antioxidant, opposingthe effect of superoxides. The antioxidant properties of NO appear toaccount for its ability to modulate proinflammatory activation ofendothelial cells. NO may also react with superoxide to formperoxynitrite which may be responsible for the cellular toxicityassociated with high levels of NO production.

[0006] NO decreases the adherence and aggregation of platelets exposedto a variety of stimuli. This has been demonstrated in vitro and in vivo(Adrie et al., Circulation 94:1919-1926 [1996]). The effect of NO onplatelet function appears to be mediated by cGMP and is augmented byPDE5 inhibitors (see PCT application WO96/25184, which is incorporatedherein by reference).

[0007] The use of NO as a drug is complicated by evidence suggestingthat high levels of NO can contribute to cell injury (Nicholson et al.,Trends Pharmacol Sci 12:19-27 [1991]). This is, at least in part,mediated by the combination of NO with reactive oxygen intermediates toform peroxynitrite which decomposes to toxic NO₂ ⁺ and OH⁻. NOcontributes to neuronal cell injury associated with cerebral ischemia(Iadecola, Trends Neurosci 20:132-139 [1997]). In addition, NO inhibitsmyocardial contractility and stimulates apoptosis of cardiac myocytes(Wu et al., J Biol Chem 272:14860-14866 [1997]), thereby impairingcardiovascular function. NO also contributes to inflammation inarthritis and possibly other autoimmune diseases when present at thesite of inflammation (Nicholson et al., Id.).

[0008] NO inhibits adherence of neutrophils to endothelium, an effectwhich may depend on mast cells (Niu et al., Circ Res 79:992-999 [1996]).

SUMMARY OF THE INVENTION

[0009] It has been discovered that inhaled gaseous nitric oxide can acton both platelets and leukocytes, affecting them in a way that leavesthem less likely to be activated once they reach a tissue susceptible toinflammation. The effect on platelets and leukocytes presumably occurswhile they are in the pulmonary circulation, since NO itself is rapidlyinactivated by hemoglobin once it contacts the blood (Rich et al., JAppl Physiol 75:1278-1284 [1993] and Rimar et al., Circulation88:2884-2887 [1993]) and so likely does not travel to distal sites ofinflammation.

[0010] Accordingly, the invention relates to a method for lessening orpreventing non-pulmonary ischemia-reperfusion injury in a mammal. Themethod includes identifying a mammal (e.g., a human) that hasischemia-reperfusion or is at risk for developing ischemia-reperfusionin a non-pulmonary tissue, and causing the mammal to inhale atherapeutically effective amount of gaseous nitric oxide. This amount issufficient to diminish the ability of circulating leukocytes orplatelets to become activated and contribute to an inflammatory processat the site of ishemia-reperfusion in the non-pulmonary tissue. Thislessens or prevents non-pulmonary ischemia-reperfusion injury in themammal. In combination with the inhaled NO gas, the mammal can beadministered a therapeutically effective amount of a second compoundthat potentiates the therapeutic effect of gaseous NO. The secondcompound can be, for example, a phosphodiesterase inhibitor (e.g.,2-o-propoxyphenyl-8-azapurin-6-one [Zaprinast™], dipyridamole,theophylline, sildenafil [Viagra™, Pfizer], or1,3-dimethyl-6-[2-propoxy-5-methanesulphonylamidophenyl]-pyrazolo[3,4-D]pyrimidin-4-[5H]-one)or superoxide dismutase. The second compound can alternatively be anantithrombotic agent such as ticlopidine, streptokinase, urokinase, t-PAor an analog thereof (e.g., met-t-PA, Retevase™, or FE1X), heparin,hirudin or an analog thereof (e.g., Hurulog™), non-steroidalanti-inflammatory agent (e.g., indomethacin or aspirin), aglucocorticoid (e.g., prednisone), or a cytotoxic agent (e.g.,methotrexate); or an anti-leukocyte agent such as an anti-leukocyteantibody.

[0011] The method is used to treat or prevent ischemia-reperfusioninjury including those caused by surgery (e.g., transplantation surgery[especially kidney or heart transplantation surgery] or heart bypasssurgery), thrombolysis, stroke, trauma-induced temporary hypotension, ora vascular interventional procedure such as atherectomy or angioplastyincluding the use of a laser, balloon, or stent. The method can be usedto treat or prevent ischemia-reperfusion injury after percutaneoustransluminal coronary angioplasty. The injury treated or prevented canoccur in any non-pulmonary tissue, including the kidney, heart, orbrain.

[0012] The invention also features a method for decreasing or preventingnon-pulmonary inflammation in a mammal. Examples of non pulmonaryinflamation are arthritis, myocarditis, encephalitis, transplantrejection, systemic lupus erythematosis, gout, dermatitis, inflammatorybowel disease, hepatitis, or thyroiditis. This method includes the stepsof identifying a mammal which has existing inflammation or is at riskfor developing inflammation in a non-pulmonary tissue; causing themammal to inhale a therapeutically effective amount of gaseous nitricoxide sufficient to diminish the ability of circulating leukocytes orplatelets to become activated in a manner that contributes to aninflammatory process in the non-pulmonary tissue, thereby decreasing orpreventing non-pulmonary inflammation in the mammal; and administeringto the mammal a therapeutically effective amount of a second compoundthat potentiates the anti-inflammatory effect of inhaled gaseous nitricoxide. The second compound can be a phosphodiesterase inhibitor (e.g.,2-o-propoxyphenyl-8-azapurin-6-one [Zaprinast™], dipyridamole,theophylline, sildenafil [Viagra™, Pfizer], or1,3-dimethyl-6-[2-propoxy-5-methanesulphonylamidophenyl]-pyrazolo[3,4-D]pyrimidin-4-[5H]-one)or superoxide dismutase. The second compound can alternatively be ananti-inflammatory drug such as a non-steroidal anti-inflammatory agent(e.g., indomethacin or aspirin), a glucocorticoid (e.g., prednisone), ora cytotoxic agent (e.g., methotrexate).

[0013] The NO gas inhaled by the mammal in the method of this inventioncan be administered at a predetermined concentration. Preferably it isadministered in the absence of tobacco smoke. Preferably thepredetermined concentration is 0.1 ppm to 300 ppm, more preferably 1 ppmto 250 ppm, and most preferably 5 ppm to 200 ppm. NO can be inhaledcontinuously or intermittently for an extended period, i.e., for atleast 24 hours.

[0014] As used herein “preventing” an injury means preventing at leastpart of the injury, and does not imply that 100% of the injury isprevented. Injury prevented is ischemia-reperfusion injury orinflammation. As used herein, injury “occurs spontaneously,” means thatthe injury has no readily observable cause.

[0015] As used herein, “potentiating the therapeutic effect of gaseousnitric oxide,” (by a second compound) means increasing the duration ormagnitude of the effect.

[0016] As used herein, “vascular interventional procedure” means anysurgical procedure that involves an anatomical disruption or amechanical disturbance of a blood vessel.

[0017] Other features and advantages of the present invention will beapparent from the following detailed description and also from theclaims.

DETAILED DESCRIPTION OF THE INVENTION

[0018] This invention relates to methods of treating or preventingischemia-reperfusion injury or inflammation through inhalation of nitricoxide gas. The methods are simple and rapid, affect non-pulmonarytissues, and do not lead to NO-associated cytotoxicity in non-pulmonarytissues.

[0019] Without further elaboration, it is believed that one skilled inthe art can, based on the above disclosure and the description below,utilize the present invention to its fullest extent. The followingdescription is to be construed as merely illustrative of how one skilledin the art can treat or prevent ischemia-reperfusion injury orinflammation in non-pulmonary tissues using inhaled nitric oxide, anddoes not limit the remainder of the disclosure in any way. Anypublications cited in this disclosure are hereby incorporated byreference.

[0020] Administration of Inhaled NO

[0021] Inhaled NO is preferably administered from a source of stored,compressed NO gas. Compressed NO gas may be obtained from a commercialsupplier such as Ohmeda, typically as a mixture of 200-800 ppm NO inpure N₂ gas. The source of NO can be 100% NO, or diluted with N₂ or anyother inert gas (e.g., helium). It is vital that the NO be obtained andstored as a mixture free of any contaminating O₂ or higher oxides ofnitrogen, because such higher oxides of nitrogen (which can form byreaction of O₂ with NO) are potentially harmful to lung tissues. Ifdesired, purity of the NO may be demonstrated with chemiluminescenceanalysis, using known methods, prior to administration to the patient.Chemiluminescence NO—NO_(x) analyzers are commercially available (e.g.,Model 14A, Thermo Environmental Instruments, Franklin, Mass.). The NO—N₂mixture may be blended with air or O₂ through, for example, calibratedrotameters which have been validated previously with a spirometer. Thefinal concentration of NO in the breathing mixture may be verified witha chemical or chemiluminescence technique well known to those in thefield (e.g., Fontijin et al., Anal Chem 42:575 [1970]). Alternatively,NO and NO₂ concentrations may be monitored by means of anelectrochemical analyzer. Any impurities such as NO₂ can be scrubbed byexposure to NaOH solutions, baralyme, or sodalime. As an additionalcontrol, the FiO₂ of the final gas mixture may also be assessed.Optionally, the ventilator can have a gas scavenger added to theexpiratory outlet to ensure that significant amounts of NO do not escapeinto the adjacent environment.

[0022] In a hospital or emergency field situation, administration of NOgas can be accomplished, for example, by attaching a tank of compressedNO gas in N₂, and a second tank of oxygen or an oxygen/N₂ mixture, to aninhaler designed to mix gas from two sources. By controlling the flow ofgas from each source, the concentration of NO inhaled by the patient canbe maintained at an optimal level. NO can also be mixed with room air,using a standard low-flow blender (e.g., Bird Blender, Palm Springs,Calif.). NO can be generated from N₂ and O₂ (i.e., air) by using anelectric NO generator. A suitable NO generator is described in Zapol,U.S. Pat. No. 5,396,882. In addition, NO can be provided intermittentlyfrom an inhaler equipped with a source of NO such as compressed NO or anelectric NO generator. The use of an inhaler may be particularlyadvantageous if a second compound (e.g., a phosphodiesterase inhibitor)is administered, orally or by inhalation, in conjunction with the NO.

[0023] NO can be administered to a mammal identified as having anon-pulmonary ischemia-reperfusion injury or inflammation, or a mammalidentified as being at risk for developing a non-pulmonaryischemia-reperfusion injury or inflammation. Preferably, the NOconcentration is 0.1 ppm to 300 ppm in air, pure oxygen, or anothersuitable gas or gas mixture. The NO can be administered for as long asneeded. The concentration can be temporarily increased for short periodsof time, e.g., 5 min at 200 ppm NO. This can be done when an immediateeffect is desired.

[0024] For treatment or prevention of non-pulmonary ischemia-reperfusioninjury or inflammation, inhaled NO can be administered by nasal prongs,mask, tent, intra-tracheal catheter or endotracheal tube, for anextended period, i.e., days or weeks. The administration can becontinuous, during the extended period. Alternatively, administrationcan be intermittent during the extended period. The administration ofgaseous NO can be via spontaneous or mechanical ventilation.

[0025] Assessment of Effects of Inhaled NO

[0026] When inhaled NO is administered, it is desirable to monitor theeffects of the NO inhalation. Such monitoring can be used, in aparticular individual, to verify desirable effects and to identifyundesirable side effects that might occur. Such monitoring is alsouseful in adjusting dose level, duration and frequency of administrationof inhaled NO in a given individual.

[0027] Other Agents Administered with NO

[0028] NO decomposes rapidly by reacting with molecular oxygen toproduce nitrite and nitrate. In addition, NO entering the blood israpidly inactivated by tight binding to hemoglobin. For these reasons,NO has only a short half-life in arterial blood. This means that inhaledNO advantageously avoids systemic vasodilation, an undesirable,potentially dangerous side effect associated with sustained systemic NOrelease from NO donor compounds such as nitroglycerin.

[0029] It may be desirable to prolong the beneficial effects of inhaledNO within leukocytes or platelets, or within cells interacting with theleukocytes or platelets in the lung. In determining how to prolong thebeneficial effects of inhaled NO, it is useful to consider that one ofthe in vivo effects of NO is activation of soluble guanylate cyclase,which stimulates production of cGMP. At least some of the beneficialeffects of NO may result from its stimulation of cGMP biosynthesis.Accordingly, in a some embodiments of the invention, a phosphodiesteraseinhibitor is administered in conjunction with NO inhalation to inhibitthe breakdown of cGMP by endogenous phosphodiesterases.

[0030] The phosphodiesterase inhibitor can be introduced into the mammalby any suitable method, including via an oral, transmucosal,intravenous, intramuscular, subcutaneous or intraperitoneal route.Alternatively, the inhibitor can be inhaled by the mammal. Forinhalation, the phosphodiesterase inhibitor is advantageously formulatedas a dry powder or an aerosolized or nebulized solution having aparticle or droplet size of less than 10 μm for optimal deposition inthe alveoli, and may optionally be inhaled in a gas containing NO.

[0031] A suitable phosphodiesterase inhibitor is Zaprinast™ (M&B 22948;2-o-propoxyphenyl-8-azapurine-6-one; Rhone-Poulenc Rorer, DagenhamEssex, UK). Zaprinast™ selectively inhibits the hydrolysis of cGMP withminimal effects on the breakdown of adenosine cyclic-monophosphate invascular smooth muscle cells (Trapani et al., J Pharmacol Exp Ther258:269 [1991]; Harris et al., J Pharmacol Exp Ther 249:394 [1989];Lugnier et al., Biochem Pharmacol 35:1743 [1986]; Souness et al., Br JPharmacol 98:725 [1989]). When using Zaprinast™ according to thisinvention, the preferred routes of administration are intravenous ororal. The suitable dose range may be determined by one of ordinary skillin the art. A stock solution of Zaprinast™ may be prepared in 0.05 NNaOH. The stock can then be diluted with Ringer's lactate solution tothe desired final Zaprinast™ concentration, immediately before use.

[0032] In a preferred embodiment, the NO is administered at 20 ppm inair for 45 min. At the start of the 45 min period, 1.0 mg of Zaprinast™per kg body weight is administered over 4 min, followed by a continuousinfusion of 0.004 mg/kg/min for the rest of the 45 min period.Alternatively, at the start of the 45 min period, 0.15 mg dipyridamoleper kg body weight is administered over 4 min, followed by a continuousinfusion of 0.004 mg/kg/min for the rest of the 45 min period. TheZaprinast™ or dipyridamole are administered in a saline solution. Inaddition, the methods are not limited to co-administration of only onedrug. For example, the administration of either phosphodiesteraseinhibitor above can be augmented by administration of a superoxidedismutase.

[0033] This invention can be practiced with other phosphodiesteraseinhibitors. Various phosphodiesterase inhibitors are known in the art,including dipyridamole and theophylline. A suitable route ofadministration and suitable dose range can be determined by one ofordinary skill in the art.

[0034] Antithrombotic agents can be administered together with NO inaccording to the invention. Such antithrombotic agents serve to (1)restore perfusion of the tissues susceptible to ischemia-reperfusioninjury via thrombolysis, and (2) augment the therapeutic effects ofinhaled NO by decreasing the potential for activiation of platelets innon-pulmonary tissues. Examples of antithrombotic agents are aspirin,streptokinase, urokinase, tissue plasminogen activator (“t-PA”),met-t-PA (i.e., t-PA with an N-terminal methionine residue), FE1X (at-PA analog), heparin, hirudin, Hirulog™ (a hirudin analog),ticlopidine, and IIb/IIIa (e.g. Rheopro™). Other antithrombotic agentscould also be used in the practice of this invention. One or more suchantithrombotic agents may be administered to a mammal before, during, orafter treatment with inhaled NO, so that the potential of platelets tobecome activated in non-pulmonary tissues is decreased.

[0035] In addition, one or more anti-leukocyte agents (e.g.,anti-leukocyte antibodies) can be administered in the methods of thisinvention. Such agents can be administered with inhaled NO with orwithout antithrombotic agents. When both anti-leukocyte agents andantithrombotic agents are administered along with NO, such agents canaugment the therapeutic effect of NO by further decreasing the potentialactivation of both leukocytes and platelets in the non-pulmonary tissuesusceptible to ischemia-reperfusion injury or inflammation.

[0036] The selection of appropriate antithrombotic and/or anti-leukocyteagents to be administered in conjunction with inhaled NO, and theselection of the appropriate dosage and route of administration of thoseantithrombotic agents, is within ordinary skill in the art.

What is claimed is:
 1. A method for lessening or preventingnon-pulmonary ischemia-reperfusion injury in a mammal, comprising thesteps of: (a) identifying a mammal that has ischemia-reperfusion or isat risk for developing ischemia-reperfusion in a non-pulmonary tissue;and (b) causing the mammal to inhale a therapeutically effective amountof gaseous nitric oxide sufficient to diminish the ability of themammal's leukocytes or platelets to become activated in a manner thatcontributes to an inflammatory process at the site of theischemia-reperfusion in the non-pulmonary tissue, thereby lessening orpreventing non-pulmonary ischemia-reperfusion injury in the mammal. 2.The method of claim 1, further comprising the step of administering tothe mammal a therapeutically effective amount of a second compound thatpotentiates the therapeutic effect of gaseous nitric oxide.
 3. Themethod of claim 2, wherein the second compound is selected from thegroup consisting of a phosphodiesterase inhibitor and superoxidedismutase.
 4. The method of claim 3, wherein the phosphodiesteraseinhibitor is selected from the group consisting of2-o-propoxyphenyl-8-azapurin-6-one, dipyridamole, theophylline,sildenafil, and1,3-dimethyl-6-(2-propoxy-5-methanesulphonylamidophenyl)-pyrazolo[3,4-D]pyrimidin-4-(5H)-one.5. The method of claim 2, wherein the second compound is selected fromthe group consisting of aspirin, ticlopidine, streptokinase, urokinase,t-PA and analogs thereof, heparin, and hirudin and analogs thereof. 6.The method of claim 1, wherein the injury is caused by surgery.
 7. Themethod of claim 6, wherein the surgery is transplantation surgery. 8.The method of claim 7, wherein the transplantation surgery is kidneytransplantation surgery or heart transplantation surgery.
 9. The methodof claim 6, wherein the surgery is heart bypass surgery.
 10. The methodof claim 1, wherein the injury is caused by a vascular interventionalprocedure.
 11. The method of claim 10, wherein the vascularinterventional procedure is angioplasty.
 12. The method of claim 11,wherein the angioplasty includes the use of a laser, balloon, or stent.13. The method of claim 11, wherein the angioplasty is an atherectomy.14. The method of claim 10, wherein the vascular interventionalprocedure is percutaneous transluminal coronary angioplasty.
 15. Themethod of claim 1, wherein the injury is caused by thrombolysis.
 16. Themethod of claim 1, wherein the injury is caused by a stroke.
 17. Themethod of claim 1, wherein the injury occurs in the kidney.
 18. Themethod of claim 1, wherein the injury occurs in the heart.
 19. Themethod of claim 1, wherein the injury occurs in the brain.
 20. Themethod of claim 1, wherein the injury occurs spontaneously.
 21. Themethod of claim 1, wherein the therapeutically effective amount ofnitric oxide is administered to the mammal at a predeterminedconcentration range.
 22. The method of claim 21, wherein theconcentration range is 0.1 ppm to 300 ppm.
 23. The method of claim 1,wherein the nitric oxide is inhaled continuously for an extended period.24. The method of claim 1, wherein the nitric oxide is inhaledintermittently for an extended period.
 25. The method of claim 1,wherein the mammal is a human.
 26. The method of claim 1, wherein theamount of gaseous nitric oxide is sufficient to diminish the ability ofplatelets to become activated in a manner that contributes to theinflammation process at the site of the ischemia-reperfusion.
 27. Amethod for decreasing or preventing non-pulmonary inflammation in amammal, comprising the steps of: (a) identifying a mammal which hasexisting inflammation or is at risk for developing inflammation in anon-pulmonary tissue; (b) causing the mammal to inhale a therapeuticallyeffective amount of gaseous nitric oxide sufficient to diminish theability of the mammal's leukocytes or platelets to become activated in amanner that contributes to an inflammation process in the non-pulmonarytissue, thereby decreasing or preventing non-pulmonary inflammation inthe mammal; and (c) immediately before, during, or after the inhalationof nitric oxide by the mammal, administering to the mammal atherapeutically effective amount of a second compound that potentiatesthe therapeutic effect of gaseous nitric oxide.
 28. The method of claim27, wherein the non-pulmonary inflammation is arthritis, myocarditis,encephalitis, transplant rejection, systemic lupus erythematosis, gout,dermatitis, inflammatory bowel disease, hepatitis, or thyroiditis. 29.The method of claim 27, wherein the second compound is selected from thegroup consisting of a phosphodiesterase inhibitor and superoxidedismutase.
 30. The method of claim 29, wherein the phosphodiesteraseinhibitor is selected from the group consisting of2-o-propoxyphenyl-8-azapurin-6-one, dipyridamole, theophylline and1,3-dimethyl-6-(2-propoxy-5-methanesulphonylamidophenyl)-pyrazolo[3,4-D]pyrimidin-4-(5H)-one.31. The method of claim 27, wherein the second compound is selected fromthe group consisting of a non-steroidal anti-inflammatory agent, aglucocorticoid, and a cytotoxic agent.
 32. The method of claim 27,wherein the nitric oxide is inhaled in a predetermined concentrationrange.
 33. The method of claim 32, wherein the concentration range is0.1 ppm to 300 ppm.
 34. The method of claim 27, wherein the nitric oxideis inhaled continuously for an extended period.
 35. The method of claim27, wherein the nitric oxide is inhaled intermittently for an extendedperiod.
 36. The method of claim 27, wherein the mammal is a human. 37.The method of claim 27, wherein the amount of gaseous nitric oxide issufficient to diminish the ability of platelets to become activated in amanner that contributes to the inflammation process.
 38. The method ofclaim 1, wherein the injury is caused by trauma.
 39. The method of claim1, wherein the injury is caused by temporary hypotension.